Spreading of antibody reactivity to non-thyroid antigens during experimental immunization with human thyroglobulin.

Intermolecular spreading of antibody reactivity has been implicated in the evolution of autoimmune disease. In this study, spreading of antibody reactivity to non-thyroid autoantigens after experimental immunization with thyroglobulin (Tg) was investigated. For this purpose, two rabbits were injected with human Tg six times (stages 1-6) every 3 weeks. Animals were also bled before priming. Antisera were tested by enzyme-linked immunosorbent assay (ELISA) for reactivity to several non-thyroid antigens: bovine serum albumin (BSA), native DNA (nDNA), human myosin, human globular (G) and filamentous (F) actin and porcine tubulin. Tg-immunized animals developed the following serological reactivity pattern: (a) high reactivity to myosin from stage 2 onward, (b) significant reactivity to F-actin, remaining high up to stage 6, (c) reactivity to BSA with a peak at stage 3, (d) a small increase of reactivity to G-actin at stage 3 and (e) no increase of reactivity to nDNA and tubulin. The study of affinity-purified anti-Tg antibodies and the use of competitive assays revealed that reactivity to F-actin was not due to cross-reaction with Tg. On the contrary, reactivity to myosin during the first stages of immunization was due to cross-reaction with Tg, while at stage 6 it became myosin-specific. Reactivity to BSA at stage 3 was also due to cross-reaction with Tg. We conclude that at least part of the induced anti-Tg antibodies may result from the expansion of B cell clones producing polyreactive natural autoantibodies, and polyreactivity of anti-Tg antibodies during the first stages of Tg-immunization may be responsible for the intermolecular spreading of antibody response.

Antibodies cross-reacting with thyroglobulin and thyroid peroxidase are induced by immunization of rabbits with an immunogenic thyroglobulin 20mer peptide.

Thyroglobulin (Tg) and thyroid peroxidase (TPO) are two major autoantigens in autoimmune thyroid diseases (AITD). Cross-reactive anti-Tg/TPO antibodies have been identified in patients with AITD and in mice immunized with Tg or TPO. In the present study, we investigated the production of anti-Tg/TPO antibodies in rabbits immunized with human Tg and with a highly immunogenic Tg peptide (namely TgP41, sequence 2651-2670 of human Tg), by noncompetitive and competitive ELISA. TgP41 was found previously to induce intramolecular epitope spreading. We found that Tg-immunized rabbits developed a serological immune response to TPO due to cross-reactivity with Tg, since serum TPO reactivity was inhibited by soluble Tg and affinity-purified anti-Tg antibodies cross-reacted with TPO. Moreover, TgP41-immunized rabbits responded to Tg and TPO. This serological response was attributed to anti-Tg/TPO antibodies, based on the observation that serum TPO reactivity was again inhibited by soluble Tg, and affinity-purified anti-Tg antibodies, induced by TgP41-immunization, cross-reacted with TPO. Purified anti-TgP41 antibodies did not react with TPO, suggesting that a putative common antigenic determinant is not included in the peptide sequence. We propose that intermolecular spreading of reactivity to TPO observed after administration of the Tg-peptide is a result of intramolecular epitope spreading to determinant(s) responsible for Tg/TPO cross-reactivity.

Evidence for intramolecular B-cell epitope spreading during experimental immunization with an immunogenic thyroglobulin peptide.

Thyroglobulin (Tg) is a target autoantigen in autoimmune thyroid diseases, such as Graves' disease (GD) and Hashimoto's thyroiditis. In a previous study we identified three 20mer Tg peptides bearing epitopes of autoantibodies associated with GD (TgP15, TgP26 and TgP41: sequences 2339-2358, 2471-2490 and 2651-2670 of human Tg, respectively). In the present study, we investigated the antigenicity of the above peptides in experimental immunization with Tg, the immunogenicity of antigenic peptides and the possibility of intramolecular B-cell epitope spreading during peptide immunization. For this purpose, two rabbits were injected with human Tg in CFA six times, every three weeks. Two control animals were injected only with CFA. Testing of antisera and of affinity-purified antibodies, by ELISA against the three peptides, revealed reactivity only to TgP41. This synthetic peptide was subsequently administered to two rabbits, in its free form (100 micro g in CFA six times, every two weeks). A strong serological response was developed not only against TgP41, but also to intact human and rabbit Tg. Immunization with TgP41 induced intramolecular B-cell epitope spreading, i.e. production of antibodies to sites on Tg other than that corresponding to TgP41, as revealed by immunoadsorption and competitive ELISA. Histopathological studies did not reveal any infiltration in thyroid glands. We conclude that peptide TgP41 encompasses not only an epitope of disease-associated autoantibodies, but also a dominant immunogenic epitope of experimentally induced Tg-specific antibodies, able to drive B-cell epitope spreading.

B-cell autoepitopes on the acetylcholinesterase-homologous region of human thyroglobulin: association with Graves' disease and thyroid eye disease.

OBJECTIVE: Thyroglobulin (Tg) is a large autoantigen involved in autoimmune thyroid diseases. Tg epitopes have, so far, been identified within large peptides. In the present study, we used small synthetic peptides to finely map serological epitopes on the highly immunogenic C-terminal region of Tg. Homology of this region to acetylcholinesterase (AChE) has been implicated in the pathogenesis of thyroid eye disease (TED) through cross-reactive antibodies. METHODS: We tested total IgG purified from four pilot Graves' disease (GD) sera reactive with both Tg and AChE and from three healthy controls, for reactivity against overlapping 20mer peptides (pin synthesis) covering the sequence 2171-2748 of human Tg. Antibody-reactive peptides were subsequently synthesized by a solid-phase technique for confirmation with a large number of sera: 99 GD, 32 Hashimoto's thyroiditis (HT) and 45 healthy controls. RESULTS: Peptides TgP15, TgP26 and TgP41 (amino acids 2339-2358, 2471-2490 and 2651-2670 respectively) were found to be targets of autoantibodies on intact Tg, recognized by a statistically significant proportion of GD sera (22.2%, 35.4% and 30.3% respectively), compared with HT (0%, 15.6% and 6.3% respectively) and healthy controls (0%, 4.4% and 4.4% respectively). The majority of GD sera (56.6%) were positive for at least one of the three peptides. In GD, TgP26 reactivity was found to be associated with TED (48.6% with TED versus 25.5% without TED, P<0.05). CONCLUSION: Some epitopes on the C-terminal region of Tg are associated with GD. A subset of Tg-reactive autoantibodies, directed to this region, is associated with TED and may be involved in the development of the disease.